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Clonal expansion analysis of transposon insertions by high-throughput sequencing identifies candidate cancer genes in a PiggyBac mutagenesis screen.

Friedel RH, Friedel CC, Bonfert T, Shi R, Rad R, Soriano P - PLoS ONE (2013)

Bottom Line: Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis.The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd).We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Department of Developmental and Regenerative Biology, Department of Neurosurgery, Icahn School of Medicine at Mount, Sinai, New York, New York, United States of America. roland.friedel@mssm.edu

ABSTRACT
Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.

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High-throughput sequencing of PiggyBac insertions.A) Kaplan-Meier survival plot of cohorts carrying either PiggyBac array alone (ATP1-S2) or PiggyBac array and constitutive transposase (ATP1-S2; R26-PBase). Red arrows indicate tumor occurrence. Tick marks denote censored animals (no tumor observed at the time of sacrifice).B) Alignments of Illumina genomic sequence reads terminate at positions generated by acoustic shearing. PB3/PB5, PiggyBac 3’/5’ terminal repeat; SA, splice acceptor; P, CAG promoter.C) Example of mapped sequence reads for the PB3 and PB5 sides of a PiggyBac insertion, viewed in the UCSC genome browser. Note that acoustic shearing causes random DNA break points to which Splinkerette adapters are ligated, which leads to a stair-like pattern of sequence alignments. The constant end of alignments to the right and left are caused by the invariable sequence read length of the Illumina system.D) Quantitative overview of sequence reads, mapped reads, and unique insertion sites from tail and tumor samples.
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pone-0072338-g001: High-throughput sequencing of PiggyBac insertions.A) Kaplan-Meier survival plot of cohorts carrying either PiggyBac array alone (ATP1-S2) or PiggyBac array and constitutive transposase (ATP1-S2; R26-PBase). Red arrows indicate tumor occurrence. Tick marks denote censored animals (no tumor observed at the time of sacrifice).B) Alignments of Illumina genomic sequence reads terminate at positions generated by acoustic shearing. PB3/PB5, PiggyBac 3’/5’ terminal repeat; SA, splice acceptor; P, CAG promoter.C) Example of mapped sequence reads for the PB3 and PB5 sides of a PiggyBac insertion, viewed in the UCSC genome browser. Note that acoustic shearing causes random DNA break points to which Splinkerette adapters are ligated, which leads to a stair-like pattern of sequence alignments. The constant end of alignments to the right and left are caused by the invariable sequence read length of the Illumina system.D) Quantitative overview of sequence reads, mapped reads, and unique insertion sites from tail and tumor samples.

Mentions: A transposon mutagenesis screen was performed in mice that carry a ubiquitously expressed PiggyBac transposase (ROSA26-PBase) and a transgenic PiggyBac transposon array (ATP1-S2) [5]. The ATP1-S2 array contains 20 copies of the transposon ATP1, which is equipped with splice acceptors for trapping of tumor suppressor genes and a CAG promoter for activation of oncogenes. These elements enable ATP1 to cause solid tumors upon transposition [5]. We bred a test cohort of 27 mice that carried ROSA26-PBase and ATP1-S2, and a control cohort of 27 mice with ATP1-S2 alone. Cohorts were aged, and while no tumors were observed in the control cohort, we observed 11 macroscopic tumors among 8 mice within the test cohort between 59 to 85 weeks (Figure 1A). Three animals carried tumors at two independent sites, and in one instance, genetic analysis of insertion sites indicated that both tumors share a common origin (see below), while all other tumors apparently arose independently. Tumor types comprised squamous cell carcinomas of the skin, solid tumors of lung and intestine, and follicular lymphoma of the spleen. The content of tumor cells in the dissected samples ranged from 30–100%, as assessed by histopathological analysis of hematoxylin and eosin stained sections (Figure S1). We isolated DNA for analysis of transposon insertion sites from all 11 tumors. To control for random insertions of PiggyBac in non-cancerous tissue, we isolated also DNA from 6 tail tips of tumor bearing mice.


Clonal expansion analysis of transposon insertions by high-throughput sequencing identifies candidate cancer genes in a PiggyBac mutagenesis screen.

Friedel RH, Friedel CC, Bonfert T, Shi R, Rad R, Soriano P - PLoS ONE (2013)

High-throughput sequencing of PiggyBac insertions.A) Kaplan-Meier survival plot of cohorts carrying either PiggyBac array alone (ATP1-S2) or PiggyBac array and constitutive transposase (ATP1-S2; R26-PBase). Red arrows indicate tumor occurrence. Tick marks denote censored animals (no tumor observed at the time of sacrifice).B) Alignments of Illumina genomic sequence reads terminate at positions generated by acoustic shearing. PB3/PB5, PiggyBac 3’/5’ terminal repeat; SA, splice acceptor; P, CAG promoter.C) Example of mapped sequence reads for the PB3 and PB5 sides of a PiggyBac insertion, viewed in the UCSC genome browser. Note that acoustic shearing causes random DNA break points to which Splinkerette adapters are ligated, which leads to a stair-like pattern of sequence alignments. The constant end of alignments to the right and left are caused by the invariable sequence read length of the Illumina system.D) Quantitative overview of sequence reads, mapped reads, and unique insertion sites from tail and tumor samples.
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Related In: Results  -  Collection

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pone-0072338-g001: High-throughput sequencing of PiggyBac insertions.A) Kaplan-Meier survival plot of cohorts carrying either PiggyBac array alone (ATP1-S2) or PiggyBac array and constitutive transposase (ATP1-S2; R26-PBase). Red arrows indicate tumor occurrence. Tick marks denote censored animals (no tumor observed at the time of sacrifice).B) Alignments of Illumina genomic sequence reads terminate at positions generated by acoustic shearing. PB3/PB5, PiggyBac 3’/5’ terminal repeat; SA, splice acceptor; P, CAG promoter.C) Example of mapped sequence reads for the PB3 and PB5 sides of a PiggyBac insertion, viewed in the UCSC genome browser. Note that acoustic shearing causes random DNA break points to which Splinkerette adapters are ligated, which leads to a stair-like pattern of sequence alignments. The constant end of alignments to the right and left are caused by the invariable sequence read length of the Illumina system.D) Quantitative overview of sequence reads, mapped reads, and unique insertion sites from tail and tumor samples.
Mentions: A transposon mutagenesis screen was performed in mice that carry a ubiquitously expressed PiggyBac transposase (ROSA26-PBase) and a transgenic PiggyBac transposon array (ATP1-S2) [5]. The ATP1-S2 array contains 20 copies of the transposon ATP1, which is equipped with splice acceptors for trapping of tumor suppressor genes and a CAG promoter for activation of oncogenes. These elements enable ATP1 to cause solid tumors upon transposition [5]. We bred a test cohort of 27 mice that carried ROSA26-PBase and ATP1-S2, and a control cohort of 27 mice with ATP1-S2 alone. Cohorts were aged, and while no tumors were observed in the control cohort, we observed 11 macroscopic tumors among 8 mice within the test cohort between 59 to 85 weeks (Figure 1A). Three animals carried tumors at two independent sites, and in one instance, genetic analysis of insertion sites indicated that both tumors share a common origin (see below), while all other tumors apparently arose independently. Tumor types comprised squamous cell carcinomas of the skin, solid tumors of lung and intestine, and follicular lymphoma of the spleen. The content of tumor cells in the dissected samples ranged from 30–100%, as assessed by histopathological analysis of hematoxylin and eosin stained sections (Figure S1). We isolated DNA for analysis of transposon insertion sites from all 11 tumors. To control for random insertions of PiggyBac in non-cancerous tissue, we isolated also DNA from 6 tail tips of tumor bearing mice.

Bottom Line: Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis.The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd).We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Department of Developmental and Regenerative Biology, Department of Neurosurgery, Icahn School of Medicine at Mount, Sinai, New York, New York, United States of America. roland.friedel@mssm.edu

ABSTRACT
Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.

Show MeSH
Related in: MedlinePlus